More than 1 in 3 people will develop cancer in their lifetime, and approximately 1,500 people die from cancer each day in the United States. Metastases, which require invasion and intravasation, are the major causes of cancer-related deaths. Key cytoskeletal, proliferative and apoptotic proteins have been shown to dynamically alter their gene expression patterns during cell invasion and intravasation, suggesting their involvement in regulating some or all of the processes involved in these cell migration phenotypes. These proteins have been termed collectively as the "Invasion Signature". One of the components of the "Invasion Signature" is Mena, a member of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family. Ena/VASP proteins are highly conserved regulators of actin dynamics, known to play critical roles in cell migration. Ena/VASP proteins contain several conserved domains that are thought to elicit specificity of Mena function through alternative splicing. Expression of a Mena isoform, MenaINV, sensitizes carcinoma cells to epidermal growth factor (EGF)- induced carcinoma cell invasion and metastasis. In response to lower levels of EGF, expression of MenaINV leads to enhanced metastasis in vivo. Furthermore, expression of MenaINV results in directional movement of carcinoma cells toward blood vessels, increased invadopodium stability, and a 200-fold increase in transendothelial migration (TEM). Expression of another Mena isoform, Mena11a, also results in increased tumor cell migration. Therefore, Mena plays critical roles in invasion and intravasation. Although many roles for Mena in tumor cell metastasis have been discovered, the mechanisms by which Mena-expressing carcinoma cells use to carry out these functions are still unknown. Thus, we will take advantage of a simplified in vitro system for specific control of parameters, as well as an in vivo system developed in our lab to take into account the tumor microenvironment. With these approaches in hand, specifically, I will test the requirement of invadopodia during Mena-induced chemotaxis, invasion and intravasation, and the upstream signaling events that regulate these processes. In particular, I propose the following aims: 1) Determining whether Mena isoform-expressing cells can autonomously chemosense a chemoattractant, and what step(s) of invadopodium formation is critical for chemosensing;2) Investigating the relationship between Mena-induced chemotaxis and invadopodium formation in vivo;and 3) Identifying the mechanism underlying the 200 fold increase in TEM activity in MenaINV-expressing cells. Ultimately, these experiments will provide insight into the mechanisms governing the observed Mena-induced metastasis in vivo, which may be critical to the development of new anti-metastatic therapies. PUBLIC HEALTH RELEVANCE: More than 1 in 3 people will develop cancer in their lifetime, and approximately 1,500 people die from cancer each day in the United States. Early identification of cancer, and knowledge about the metastatic potential of a tumor may facilitate treatment, and thus increase survival. With this work, we hope to understand the molecular basis of tumor cell spread, with the goal of uncovering new markers to identify high-risk patients and ultimately develop new anti-metastatic therapies.